Members of the ligand-induced nuclear receptor superfamily, including steroid receptors are medically important regulators of cellular activity. Hormone activated nuclear receptors regulate transcription of their cognate target genes by binding to specific DNA recognition sites. Recently, receptors were shown to be highly regulated in their subcellular distribution. Presumably, the ligand-induced activation process includes receptor redistribution between cytoplasm and nucleus and within the nucleus. The development of chimeras between fluorescent proteins and the nuclear receptors provides a new approach to address the roles of receptor trafficking in hormone actions. In collaboration with Dr. Hager's laboratory we explored glucocorticoid receptor functions with a green fluorescent protein chimera of the rat glucocorticoid receptor (GFP-GR). Transient expression of GFP-GR in mouse adenocarcinoma cells allowed us to study receptor motion and intranuclear targeting. We demonstrated that unliganded GR resides in the cytoplasm and that hormone addition initiates a cytoplasm to nucleus translocation. We detected distinct subnuclear localizations of agonist (dexamethasone) and antagonist (RU486) activated receptors. After dexamethasone, GFP-GR accumulated in a discrete series of foci, while after RU486 GFP-GR did not forming bright foci but remained in a reticular pattern. We continued to use GFP-GR plasmid from Dr. Hager's laboratory and also began using a plasmid from Dr. Chrousos's laboratory containing a GFP-chimera of the human GR, to study receptor translocation and intranuclear targeting. Direct observation of GFP-GR binding to biologically relevant regulatory sites in chromatin was visualized in an adenocarcinoma cell line (904.13). These cells carry a "minichromosome", a highly amplified set of mouse mammary tumor virus reporter element that contains 800 GR binding sites in a head-to-tail tandem repeat. Agonist exposure caused GFP-GR accumulation along this array. We recently calculated GFP-GR motion rates within the nucleus and residence time on target genes with fluorescence recovery after photobleaching. These studies showed that after dexamethasone, receptor binds to target sites for less than a minute. We generated transcriptionally inactive DNA-binding zinc-finger mutants of GFP-GR. These mutant receptors translocated into the nucleus after dexamethasone addition, but remained in fast motion within the nucleus and failed to bind to the "minichromosome". We will continue to study the subcellular and subnuclear distribution of the receptor family members to define protein-protein interactions that regulate receptor motion and to address important questions regarding selective hormone actions.